(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property Organization
International Bureau
(43) International Publication Date VC^T International Publication Number
19 June 2008 (19.06.2008) WO 2008/071394 Al
(51) International Patent Classification:
C07K 16/18 (2006.01) A61K 9/00 (2006.01)
A61K 39/395 (2006.01) A61K 47/00 (2006.01)
(21) International Application Number:
PCT/EP2007/0 10825
(22) International Filing Date:
1 1 December 2007 (11.1 2.2007)
(25) Filing Language: English
(26) Publication Language: English
(30) Priority Data:
06025590.8 11 December 2006 (1 1.12.2006) EP
(71) Applicant (for all designated States except US): F. HOFF-
MANN-LA ROCHE AG [CH/CH]; Grenzacherstrasse
124, CH-4070 Basel (CH).
(72) Inventors; and
(75) Inventors/Applicants (for US only): GOLDBACH,
Pierre [FR/FR]; 7, rue des Fleurs, F-68170 Rixheim (FR).
MAHLER, Hanns-Christian [DE/CH] ; Riehenring 15,
CH-4058 Basel (CH). MUELLER, Robert [DE/CH];
Haltingerstrasse 29, CH-4057 Basel (CH). WURTH,
Christine [LU/DE]; Adlergaesschen 6, 79539 Loerrach
(DE).
(74) Agent: VOSSIUS & PARTNER; Siebertstrasse 4, 81675
Munich (DE).
(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL,
IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL,
PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
ZM, ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
Published:
— with international search report
— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments
— with sequence listing part of description published sepa-
rately in electronic form and available upon request from
the International Bureau
(54) Title: ABETA ANTIBODY PARENTERAL FORMULATION
(57) Abstract: The present invention relates to a stable pharmaceutical parenteral formulation of an antibody, antibody molecule, a
mixture of antibodies and/or a mixture of antibody molecules against the amyloid4>eta peptide (Abeta) and a process for the prepa-
ration. Furthermore, corresponding uses are described.
WO 2008/071394
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Abeta antibody parenteral formulation
The present invention relates to a stable pharmaceutical parenteral formulation of an
antibody, antibody molecule, a mixture of antibodies and/or a mixture of antibody molecules
against the amyloid-beta peptide (Abeta) and a process for the preparation thereof.
Furthermore, corresponding uses are described.
In a first aspect, the invention relates to a stable pharmaceutical parenteral Abeta
antibody pharmaceutical formulation comprising:
about 1 to about 250 mg/mL Abeta antibody;
about 0.001 to about 1% of at least one surfactant;
about 1 to about 100 mM of a buffer;
optionally about 10 to about 500 mM of a stabilizer and/or about 5 to about 500 mM
of a tonicity agent;
at a pH of about 4.0 to about 7.0.
In particular, the present invention relates to an Abeta antibody formulation wherein
the comprised Abeta antibodies (or mixtures thereof) are capable of specifically binding the
amyloid-beta peptide. Antibodies that specifically bind Abeta are known in the art. Specific
examples of Abeta antibody that can be used in the formulation according to the invention
have been described in the published PCT patent application WO 03/070760 and especially
in the claims, the content of which is incorporated herein by reference.
The amyloid-beta peptide, which is also termed "amyloid (3", "Ap", "Ap4" or "p-A4"
and, in particular in context of this invention "Abeta", is a main component of the
extracellular neuritic plaques that are associated with amyloidogenic diseases such as
Alzheimer's disease; see Selkoe (1994), Ann. Rev. Cell Biol. 10, 373-403, Koo (1999),
PNAS Vol. 96, pp. 9989-9990, US 4,666,829 or Glenner (1984), BBRC 12, 1131. This
amyloid p is derived from "Alzheimer precursor protein/p-amyloid precursor protein" (APP).
APPs are integral membrane glycoproteins (see Sisodia (1992), PNAS Vol. 89, pp. 6075)
and are endoproteolytically cleaved within the Abeta sequence by a plasma membrane
protease, a-secretase (see Sisodia (1992), loc. cit.). Furthermore, further secretase activity, in
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particular (3-secretase and y-secretase activity leads to the extracellular release of amyloid-P
(AP) comprising either 39 amino acids (AP39), 40 amino acids (AP40), 42 amino acids
(AP42) or 43 amino acids (Ap43); see Sinha (1999), PNAS 96, 11094-1053; Price (1998),
Science 282, 1078 to 1083; WO 00/72880 or Hardy (1997), TINS 20, 154.
Ap has several naturally occurring forms, whereby the human forms are referred to as
the above mentioned Ap39, AP40, AP41, AP42 and AP43. The most prominent form, AP42,
has the amino acid sequence (starting from the N-terminus):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3). In
AP41, AP40, AP39, the C-terminal amino acids A, IA and VIA are missing, respectively. In
the Ap43-form an additional threonine residue is comprised at the C-terminus of the above
depicted sequence (SEQ ID NO: 3).
Antibody molecules, as part of the group of protein pharmaceuticals, are very
susceptible to physical and chemical degradation, such as denaturation and aggregation,
deamidation, oxidation and hydrolysis. Protein stability is influenced by the characteristics of
the protein itself, e.g. the amino acid sequence, and by external influences, such as
temperature, solvent pH, excipients, interfaces, or shear rates. So, it is important to define the
optimal formulation conditions to protect the protein against degradation reactions during
manufacturing, storage and administration. (Manning, M. C., K. Patel, et al. (1989).
"Stability of protein pharmaceuticals." Pharm Res 6(1 1): 903-18., Zheng, J. Y. and L. J. Janis
(2005). "Influence of pH, buffer species, and storage temperature on physicochemical
stability of a humanized monoclonal antibody LA298." Int_J_Pharm.)
Administration of antibodies via subcutaneous or intramuscular route requires high
protein concentration in the final formulation due to the often required high doses and the
limited administration volumes. (Shire, S. J., Z. Shahrokh, et al. (2004). "Challenges in the
development of high protein concentration formulations." J Pharm Sci 93(6): 1390-402.,
Roskos, L. K., C. G. Davis, et al. (2004). "The clinical pharmacology of therapeutic
monoclonal antibodies." Drug Development Research 61(3): 108-120.) The large-scale
manufacturing of high protein concentration can be achieved by ultrafiltration processes,
drying process, such as lyophilisation or spray-drying, and precipitation processes. (Shire, S.
J., Z. Shahrokh, et al. (2004). "Challenges in the development of high protein concentration
formulations." J Pharm Sci 93(6): 1390-402.)
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Andya et al. (US patent 6,267,958, US patent 6,85,940) describe a stable lyophilized
formulation of an antibody, which is reconstituted with a suitable diluent volume to achieve
the required concentration. The formulation comprises a lyoprotectant, a buffer and a
surfactant.
Liu et al. (Liu, J., M. D. Nguyen, et al. (2005). "Reversible self-association increases
the viscosity of a concentrated monoclonal antibody in aqueous solution." J Pharm Sci 94(9):
1928-40.) examined the viscosity behavior of high concentration antibody formulations.
Three monoclonal antibodies, constructed from the identical IgGl framework, were
examined for their self-association at high protein concentration. The three antibodies
demonstrated no consistent viscosity-profile and showed significant differences in their self-
association behavior.
One object of the present invention is to provide a formulation of an Abeta antibody or
of mixtures of such antibodies, which is/are concentrated to the required concentration by
reconstitution of a lyophilized formulation with a suitable volume or by removing the solvent
by an ultrafiltration process. The formulation demonstrates sufficient stability during
manufacturing, storage and administration. As demonstrated by Liu et al., antibodies show an
unpredictable viscosity-concentration profile. (Liu, J., M. D. Nguyen, et al. (2005).
"Reversible self-association increases the viscosity of a concentrated monoclonal antibody in
aqueous solution." J Pharm Sci 94(9): 1928-40.) In comparison to the patents US 6,267,958
and US 6,685,940 the presented formulation provides equal or better stability of an Abeta
human antibody during storage and has a viscosity, which is suitable for the subcutaneous or
intramuscular administration route.
Examples of Abeta antibodies that are useful in the present invention are
immunoglobulin molecules, e.g. IgG molecules. IgGs are characterized in comprising two
heavy and two light chains (illustrated e.g. in figure 1) and these molecules comprise two
antigen binding sites. Said antigen binding sites comprise "variable regions" consisting of
parts of the heavy chains (VH) and parts of the light chains (VL). The antigen-binding sites
are formed by the juxtaposition of the VH and VL domains. For general information on
antibody molecules or immunoglobulin molecules see also common textbooks, like Abbas
"Cellular and Molecular Immunology", W.B. Sounders Company (2003).
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In one embodiment, the parenteral formulation of the present invention comprises
Abeta antibody (or mixture of such antibodies) in which in at least one of the variable
regions in the heavy chain of said antibodies comprises a N-glycosylation. The glycosylated
asparagine (Asn) in the variable region of the heavy chain (VH) may be in the
complementarity determining region 2 (CDR2 region), said glycosylated asparagine (Asn)
may be on position 52 in the variable region of the heavy chain (VH) as shown in SEQ ID
NO: 1.
The term "mono-glycosylated antibody" relates to an antibody molecule comprising an
N-glycosylation in one (V H )-region of an individual antibody molecule"; see also figure 1 .
The term "double-glycosylation antibody" defines an antibody molecule which is N-
glycosylated on both variable regions of the heavy chain" (figure 1). Antibody molecules
which lack a N-glycosylation on both heavy chain (VH)-domains are named "non-
glycosylated antibodies" (figure 1). The mono-glycosylated antibody, the double-
glycosylated antibody and the non-glycosylated antibody may comprise the identical amino
acid sequences or different amino acid sequences.
The mono-glycosylated antibody and the double-glycosylated antibody are herein
referred to as "glycosylated antibody isoforms". A purified antibody molecule characterized
in that at least one antigen binding site comprises a glycosylation in the variable region of the
heavy chain (VH) is a mono-glycosylated antibody which is free of or to a very low extent
associated with an isoform selected from a double-glycosylated antibody and a non-
glycosylated antibody, i.e. a "purified mono-glycosylated antibody". A double-glycosylated
antibody in context of this invention is free of or to a very low extent associated with an
isoform selected from a mono-glycosylated antibody and a non-glycosylated antibody, i.e. a
"purified double-glycosylated antibody".
The formulations according to this invention may contain mono-glycosylated or
double-glycosylated or non-glycosylated antibodies, or specifically defined mixtures thereof.
The antibody mixtures or antibody pools provided herein may comprise 50% mono-
glycosylated and 50% double-glycosylated antibodies as defined herein. However, also
envisaged are the ratios of 30/70 to 70/30. Yet, the person skilled in the art is aware that also
other ratios are envisaged in the antibody mixtures of this invention. For example, also 10/90
or 90/10, 20/80 or 80/20 as well as 40/60 or 60/40 may be employed in context of this
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invention. A particular useful ratio in the antibody mixtures comprised in the formulation of
the invention comprises double-glycosylated and mono-glycosylated antibody as defined
herein above is a ratio from 40/60 to 45/55.
The term "which is free of or to a very low extent" denotes the complete absence of the
respective other (glycosylation) isoforms or a presence of another (glycosylated) isoform in a
concentration of at the most 1 0 %, e.g. at the most 5%, e.g. at the most 4%, e.g. at the most
3%, e.g. at the most 2%, e.g. at the most 1%, e.g. at the most 0.5%, e.g. at the most 0.3%,
e.g. at the most 0.2%.
The term "antibody(ies)" is used herein synonymously with the term "antibody
molecule(s)" and comprises, in the context of the present invention, antibody molecule(s)
like full immunoglobulin molecules, e.g. IgMs, IgDs, IgEs, IgAs or IgGs, like IgGl, IgG2,
IgG2b, IgG3 or IgG4 as well as to parts of such immunoglobulin molecules, like Fab-
fragments, Fab '-fragments, F(ab)2-fragements, chimeric F(ab)2 or chimeric Fab' fragments,
chimeric Fab-fragments or isolated VH- or CDR-regions (said isolated VH- or CDR-regions
being, e.g. to be integrated or engineered in corresponding "framework(s)") Accordingly, the
term "antibody" also comprises known isoforms and modifications of immunoglobulins, like
single-chain antibodies or single chain Fv fragments (scAB/scFv) or bispecific antibody
constructs, said isoforms and modifications being characterized as comprising at least one
glycosylated VH region as defined herein. A specific example of such an isoform or
modification may be a sc (single chain) antibody in the format VH-VL or VL-VH, wherein
said VH comprises the herein described glycosylation. Also bispecific scFvs are envisaged,
e.g. in the format VH-VL- VH-VL, VL-VH- VH-VL, VH-VL- VL-VH. Also comprised in the
term "antibody" are diabodies and molecules that comprise an antibody Fc domain as a
vehicle attached to at least one antigen binding moiety/peptide, e.g. peptibodies as described
in WO 00/24782. It is evident from the above that the present invention also relates to
parenteral formulations of Abeta antibodies that comprise "mixtures" of antibodies/antibody
molecules. A particular "mixture" of said antibodies is described above, namely a mixture of
"mono" and "double"-glycosylated antibodies directed against Abeta.
"Antibody fragments" also comprises such fragments which per se are not able to
provide effector functions (ADCC/CDC) but provide this function in a manner according to
the invention after being combined with appropriate antibody constant domain(s).
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The Abeta antibody(ies) that may be comprised in the inventive formulation(s) are,
inter alia, recombinantly produced Abeta antibody(ies). These may be produced in a
mammalian cell-culture system, e.g. in CHO cells. Such mammalian cell culture systems are
particular useful in the preparation of Abeta antibodies or Abeta antibodies/antibody
molecules that are glycosylated like the specific herein exemplified Abeta antibody that
comprises a N-glycosylation in the variable region. The antibody molecules may be further
purified by a sequence of chromatographic and filtration steps e.g. in order to purify the
specific glycosylated antibody isoforms as described herein below.
The terms "monoclonal antibody" or "monoclonal antibody composition" as used
herein refer to a preparation of antibody molecules of a single amino acid composition.
Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single
binding specificity which have variable and constant regions derived from human germline
immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are
produced by a hybridoma which includes a B cell obtained from a transgenic non-human
animal, e.g. a transgenic mouse, having a genome comprising a human heavy chain transgene
and a light human chain transgene fused to an immortalized cell.
The term "chimeric antibody" refers to a monoclonal antibody comprising a variable
region, i.e., binding region, from one source or species and at least a portion of a constant
region derived from a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region and a human constant
region are especially preferred. Such murine/human chimeric antibodies are the product of
expressed immunoglobulin genes comprising DNA segments encoding murine
immunoglobulin variable regions and DNA segments encoding human immunoglobulin
constant regions. Other forms of "chimeric antibodies" encompassed by the present invention
are those in which the class or subclass has been modified or changed from that of the
original antibody. Such "chimeric" antibodies are also referred to as "class-switched
antibodies." Methods for producing chimeric antibodies involve conventional recombinant
DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S.L.,
et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and
5,204,244.
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The term "humanized antibody" refers to antibodies in which the framework or
"complementarity determining regions" (CDR) have been modified to comprise the CDR of
an immunoglobulin of different specificity as compared to that of the parent
immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody." See, e.g., Riechmann, L.,
et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270.
Particularly preferred CDRs correspond to those representing sequences recognizing the
antigens noted above for chimeric and bifunctional antibodies.
The term "human antibody", as used herein, is intended to include antibodies having
variable and constant regions derived from human germline immunoglobulin sequences. The
variable heavy chain is preferably derived from germline sequence DP-50 (GenBank
L06618) and the variable light chain is preferably derived from germline sequence L6
(GenBank X01668). The constant regions of the antibody are constant regions of human
IgGl type. Such regions can be allotypic and are described by, e.g., Johnson, G., and Wu,
T.T., Nucleic Acids Res. 28 (2000) 214-218 and the databases referenced therein and are
useful as long as the properties of induction of ADCC and preferably CDC according to the
invention are retained.
The term "recombinant human antibody", as used herein, is intended to include all
human antibodies that are prepared, expressed, created or isolated by recombinant means,
such as antibodies isolated from a host cell such as an SP2-0, NS0 or CHO cell (like CHO
Kl) or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or
antibodies expressed using a recombinant expression vector transfected into a host cell. Such
recombinant human antibodies have variable and constant regions derived from human
germline immunoglobulin sequences in a rearranged form. The recombinant human
antibodies according to the invention have been subjected to in vivo somatic hypermutation.
Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are
sequences that, while derived from and related to human germline VH and VL sequences,
may not naturally exist within the human antibody germline repertoire in vivo.
As used herein, "binding" refers to antibody binding to Abeta with an affinity of about
10 -13 to 10 -8 M (K D ), preferably of about 10 -13 to 10* 9 M.
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The "constant domains" are not involved directly in binding the antibody to an antigen
but are involved in the effector functions (ADCC, complement binding, and CDC). The
constant domain of an antibody according to the invention is of the IgGl type. Human
constant domains having these characteristics are described in detail by Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, MD. (1991), and by Bruggemann, M., et al., J. Exp. Med. 166
(1987) 1351-1361; Love, T.W., et al., Methods Enzymol. 178 (1989) 515-527. Examples are
shown in SEQ ID NOs: 5 to 8 in WO 2005/005635. Other useful and preferred constant
domains are the constant domains of the antibodies obtainable from the hybridoma cell lines
deposited with depositories like DSMZ or ATCC. The constant domains may provide
complement binding. ADCC and optionally CDC are provided by the combination of
variable and constant domains.
The "variable region" (variable region of a light chain (VL), variable region of a heavy
chain (VH)) as used herein denotes each of the pair of light and heavy chains which is
involved directly in binding the antibody to the antigen. The domains of variable human light
and heavy chains have the same general structure and each domain comprises four
framework (FR) regions whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions, CDRs). The framework
regions adopt a p-sheet conformation and the CDRs may form loops connecting the p-sheet
structure. The CDRs in each chain are held in their three-dimensional structure by the
framework regions and form together with the CDRs from the other chain the antigen
binding site. The antibody heavy and light chain CDR3 regions play a particularly important
role in the binding specificity/affinity of the antibodies according to the invention and
therefore provide a further object of the invention.
The terms "hypervariable region" or "antigen-binding portion of an antibody" when
used herein refer to the amino acid residues of an antibody which are responsible for antigen-
binding. The hypervariable region comprises amino acid residues from the "complementarity
determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain
regions other than the hypervariable region residues as herein defined. Therefore, the light
and heavy chains of an antibody comprise from N- to C-terminus the domains FR1, CDR1,
FR2, CDR2, FR3, CDR3, and FR4. Especially, CDR3 of the heavy chain is the region which
contributes most to antigen binding. CDR and FR regions are determined according to the
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standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those
residues from a "hypervariable loop".
The formulation of this invention may, inter alia, comprise "stabilizers",
"lyoprotectants", "sugars", "amino acids", "polyols", "antioxidants", "preservatives",
"surfactants", "buffers" and/or "tonicity agents".
The term "stabilizer" denotes a pharmaceutical acceptable excipient, which protects the
active pharmaceutical ingredient and/ or the formulation from chemical and / or physical
degradation during manufacturing, storage and application. Chemical and physical
degradation pathways of protein pharmaceuticals are reviewed by Cleland, J. L., M. F.
Powell, et al. (1993). "The development of stable protein formulations: a close look at
protein aggregation, deamidation, and oxidation." Crit Rev Ther Drug Carrier Syst 10(4):
307-77, Wang, W. (1999). "Instability, stabilization, and formulation of liquid protein
pharmaceuticals." Int J Pharm 185(2): 129-88., Wang, W. (2000). "Lyophilization and
development of solid protein pharmaceuticals." Int J Pharm 203(1-2): 1-60. and Chi, E. Y.,
S. Krishnan, et al. (2003). "Physical stability of proteins in aqueous solution: mechanism and
driving forces in normative protein aggregation." Pharm Res 20(9): 1325-36. Stabilizers
include but are not limited to sugars, amino acids, polyols, surfactants, antioxidants,
preservatives, cyclodextrines, e.g. hydroxypropyl-(3-cyclodextrine, sulfobutylethyl-p-
cyclodextrin, p-Cyclodextrin, polyethylenglycols, e.g. PEG 3000, 3350, 4000, 6000,
albumin, e.g. human serum albumin (HSA), bovines serum albumin (BSA), salts, e.g.
sodium chloride, magnesium chloride, calcium chloride, chelators, e.g. EDTA as hereafter
defined. As mentioned hereinabove, stabilizers can be present in the formulation in an
amount of about 10 to about 500 mM, preferably in an amount of about 10 to about 300mM
and more preferably in an amount of about 1 OOmM to about 300mM.
The term "lyoprotectant" denotes pharmaceutical acceptable excipients, which protects
the labile active ingredient (e.g. a protein) against destabilizing conditions during the
lyophilisation process, subsequent storage and reconstitution. Lyoprotectants comprise but
are not limited to the group consisting of sugars, polyols (such as e.g. sugar alcohols) and
amino acids. Preferred lyoprotectants can be selected from the group consisting of: sugars
such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose,
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and raffinose neuraminic acid and galactosamine, amino sugars such as glucosamine, N-
Methylglucosamine ("Meglumine"), polyols such as mannitol, and amino acids such as
arginine. Lyoprotectants are generally used in an amount of about 10 to 500mM, preferably
in an amount of about 1 0 to about 300mM and more preferably in an amount of about 100 to
about 300mM.
The term "sugar" as used herein denotes a pharmaceutically acceptable carbohydrate
used generally in an amount of about 10 mM to about 500 mM, preferably in an amount of
about 10 to about 300mM and more preferably in an amount of about 100 to about 300mM.
Suitable sugars comprise but are not limited to trehalose, sucrose, lactose, glucose, mannose,
maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-Methylglucosamine (so-
called "Meglumine"), galactosamine and neuraminic acid. Preferred sugars are sucrose and
trehalose and more preferably sucrose.
The term "amino acid" as used herein in the context of the pharmaceutical parenteral
formulation denotes a pharmaceutical acceptable organic molecule possessing an amino
moiety located at a-position to a carboxylic group. Amino acids comprise but not limited to
arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine,
alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations
thereof. Amino acids are generally used in an amount of about 10 to 500mM, preferably in
an amount of about 10 to about 300mM and more preferably in an amount of about 100 to
about 300mM.
The term "polyols" as used herein denotes pharmaceutically acceptable alcohols with
more than one hydroxy group. Polyols can be used in an amount of about 10 mM to about
500mM, preferably in an amount of about 10 to about 300 and more preferably in an amount
of about 100 to about 300mM. Suitable polyols comprise to but are not limited to mannitol,
sorbitol, glycerine, dextran, glycerol, arabitol, propylene glycol, polyethylene glycol, and
combinations thereof.
The term "antioxidant" denotes pharmaceutically acceptable excipients, which prevent
oxidation of the active pharmaceutical ingredient. Antioxidants can be used in an amount of
about 1 to about lOOmM, preferably in an amount of about 5 to about 50mM and more
preferably in an amount of about 5 to about 20mM. Antioxidants comprise but are not
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limited to ascorbic acid, glutathione, cysteine, methionine, citric acid, EDTA, and
combinations thereof.
The term "preservative" denotes pharmaceutically acceptable excipients, which prevent
the growth of microorganism in the formulation. For example, the addition of a preservative
to a multi-dose formulation protects the formulation against microbial contamination.
Preservatives are generally used in an amount of about 0.001 to about 2 %(w/v).
Preservatives comprise but are not limited to ethanol, benzyl alcohol, phenol, m-cresol, p-
chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations
thereof.
The term "surfactant" as used herein denotes a pharmaceutically acceptable surfactant.
In the formulation of the invention, the amount of surfactant is described a percentage
expressed in weight/volume percent (w/v %). Suitable pharmaceutically acceptable
surfactants comprise but are not limited to the group of polyoxyethylensorbitan fatty acid
esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers
(Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic)., and
sodium dodecyl sulphate (SDS). Preferred polyoxyethylenesorbitan-fatty acid esters are
polysorbate 20,(sold under the trademark Tween 20™) and polysorbate 80 (sold under the
trademark Tween 80™). Preferred polyethylene-polypropylene copolymers are those sold
under the names Pluronic® F68 or Poloxamer 1 88™. Preferred Polyoxyethylene alkyl ethers
are those sold under the trademark Brij™. Preferred alkylphenolpolyoxyethylene ethers are
sold under the tradename Triton-X. When polysorbate 20 (Tween 20™) and polysorbate
80(Tween 80™) are used they are generally used in a concentration range of about 0.001 to
about 1%, preferably of about 0.005 to about 0.1% and still preferably about 0.01% to about
0.04%w/v.
The term "buffer" as used herein denotes a pharmaceutically acceptable excipient,
which stabilizes the pH of a pharmaceutical preparation. Suitable buffers are well known in
the art and can be found in the literature. Preferred pharmaceutically acceptable buffers
comprise but are not limited to histidine-buffers, citrate-buffers, succinate-buffers and
phosphate-buffers. Still preferred buffers comprise L-histidine or mixtures of L-histidine and
L-histidine hydrochloride with pH adjustment with an acid or a base known in the art. The
abovementioned histidine-buffers are generally used in an amount of about ImM to about
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100 mM, preferably of about 5 mM to about 50 mM and still more preferably of about 10-20
mM. Independently from the buffer used, the pH can be adjusted at a value comprising about
4.0 to about 7.0 and preferably about 5.0 to about 6.0 and still preferably about 5.5 with an
acid or a base known in the art, e.g., hydrochloric acid, acetic acid, phosphoric acid, sulfuric
acid and citric acid, sodium hydroxide and potassium hydroxide
The term "tonicity agents" as used herein denotes pharmaceutically acceptable tonicity
agents. Tonicity agents are used to modulate the tonicity of the formulation. The formulation
can be hypotonic, isotonic or hypertonic. Isotonicity is generally relates to the osmotic
pressure relative of a solution usually relative to that of human blood serum. The formulation
according to the invention can be hypotonic, isotonic or hypertonic but will preferably be
isotonic. In a concern for clarity it is once more emphasized that an isotonic formulation is
liquid or liquid reconstituted from a solid form, e.g. from a lyophilized form and denotes a
solution having the same tonicity as some other solution with which it is compared, such as
physiologic salt solution and the blood serum. Suitable isotonicity agents comprise but are
not limited to sodium chloride, potassium chloride, glycerin and any component from the
group of amino acids, sugars, in particular glucose as defined herein as well as combinations
thereof. Tonicity agents are used in an amount of about 5 mM to about 500 mM.
The term "liquid" as used herein in connection with the formulation according to the
invention denotes a formulation which is liquid at a temperature of at least about 2 to about 8
°C under standard pressure.
The term "lyophilizate" as used herein in connection with the formulation according to
the invention denotes a formulation which is manufactured by freeze-drying methods known
in the art per se. The solvent (e.g. water) is removed by freezing following sublimation under
vacuum and desorption of residual water at elevated temperature. In the pharmaceutical field,
the lyophilizate has usually a residual moisture of about 0.1 to 5% (w/w) and is present as a
powder or a physical stable cake. The lyophilizate is characterized by a fast dissolution after
addition of a reconstitution medium.
The term "reconstituted formulation" as used herein in connection with the formulation
according to the invention denotes a formulation which is lyophilized and re-dissolved by
addition of reconstitution medium. The reconstitution medium comprises but is not limited
to water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride
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solutions (e.g. 0.9% (w/v) NaCl), glucose solutions (e.g. 5% glucose), surfactant containing
solutions (e.g. 0.01% polysorbate 20), a pH -buffered solution (e.g. phosphate-buffered
solutions) and combinations thereof.
The term "stable formulation" as used herein in connection with the formulation
according to the invention denotes a formulation, which preserves its physical and chemical
integrity during manufacturing, storage and application. Various analytical techniques for
evaluating protein stability are available and reviewed in Reubsaet, J. L., J. H. Beijnen, et al.
(1998). "Analytical techniques used to study the degradation of proteins and peptides:
chemical instability". J Pharm Biomed Anal 17(6-7): 955-78 and Wang, W. (1999).
"Instability, stabilization, and formulation of liquid protein pharmaceuticals." Int J Pharm
185(2): 129-88. Stability can be evaluated by storage at selected climate conditions for a
selected time period, by applying mechanical stress such as shaking at a selected shaking
frequency for a selected time period, by irradiation with a selected light intensity for a
selected period of time, or by repetitive freezing and thawing at selected temperatures.
The term "pharmaceutically acceptable" as used herein in connection with the
formulation according to the invention denotes a formulation which is in compliance with
the current international regulatory requirements for pharmaceuticals. A pharmaceutical
acceptable formulation contains excipients which are generally recognized for the anticipated
route of application and concentration range as safe. In addition, it should provide sufficient
stability during manufacturing, storage and application. Especially a formulation for a
parenteral route of application should fulfill the requirements isotonicity and euhydric pH in
comparison to the composition of human blood.
As mentioned above, in one aspect, the invention relates to a stable pharmaceutical
parenteral Abeta antibody formulation comprising:
about 1 to about 250 mg/mL Abeta antibody;
about 0.001 to about 1% of at least one surfactant;
about 1 to about 100 mM of a buffer;
- optionally about 1 0 to about 500 mM of a stabilizer and/or about 5 to about 500 mM
of a tonicity agent
- at a pH of about 4.0 to about 7.0.
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The Abeta antibody concentration ranges from about 1 to about 250 mg/mL, preferably
from about 50 mg/mL to about 200 mg/mL and more preferably from about 150 mg/mL to
about 200 mg/mL. For clarity reasons, it is emphasized that the concentrations as indicated
herein relate to the concentration in a liquid or in a liquid that is accurately reconstituted
from a solid form. Accordingly, the lyophilized formulations as described herein can be
reconstituted from a lyophilizate in such way that the resulting reconstituted formula
comprises the respective constituents in the concentrations described herein.
However, it is evident for the skilled person that the stable lyophilizates as described
herein may also be reconstituted using such an amount of reconstitution medium that the
resulting reconstituted formulation is either more concentrated or less concentrated. For
instance, the lyophilizate of "Formulation A" as described herein in Table 2 may be
reconstituted in such way that the resulting reconstituted formulation is further diluted to
comprise e.g. 20mg/mL Abeta antibody, 5.3mM L-histidine, 66. 7mM Sucrose and 0.011%
polysorbate 20; see Formulation R of Table 2.
The formulation according to the invention can be in a liquid form, a lyophilized form
or in a liquid form reconstituted from a lyophilized form.
In the cases where the formulation of the invention is in a lyophilized form or in a
liquid from reconstituted from a lyophilized form, it can comprise at least one lyoprotectant
as stabilizer.
The formulation according to the invention can be administered by intravenous (i.v.),
subcutaneous (s.c.) or any other parenteral administration means such as those known in the
pharmaceutical art. The formulation according to the invention is preferably administered by
subcutaneous ways.
The formulation according to the invention can be prepared by methods known in the
art, such as ultrafiltration-diafiltration, dialysis, addition and mixing, lyophilisation,
reconstitution, and combinations thereof. Examples of preparations of formulations
according to the invention can be found hereinafter.
In a preferred embodiment, the Abeta antibody comprised in the pharmaceutical
parenteral formulation of the present invention may comprise or have the variable region as
defined in SEQ ID NO: 1:
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QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGT
RTYYADSVKGPvFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVR
YFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL
SLSPGK (SEQ ID NO: 1)
This sequence is also depicted herein below and the CDRs, CH-regions, heavy regions
as well as two N-glycosylation sites (Asn 52 and Asn 306) are indicated:
QVELVESGGGLVQPGGSLRLSCAAS |GFTFSSYAMS| WVRQAPGKGLEWVS
ArNASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
GKGNTHKPYGYVRYFDV| WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTOTYICNV
-NHKPSNTKVDKKV EPKSCDKTHTCPPCP APFJ J ,GGPS VF LFPPKPKD TLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGV EVHNAK TKPREEOYNSTYRVVSVLTVLHODWL
NGKEYKCK VSNKALPAPTEKTTSKAKGOPREPOVYTLPPSRDELTKNOVSLTCLVKG
FYPSP^yE^^SNGQPENNYKTTP
MHE ALIFNH YXQK.§LS L S P.GK .(SEQ ID NO: 1)
framed| :CDRl, 2, 3
underlined : CHI
italics: hinge
underlined twice : CH2
dotted underlined:, CH3
bold N: N-linked glycosylation sites
The exemplified Abeta antibody comprising SEQ ID NO: 1 as described herein may
also comprise a light chain, said light chain may comprise or have the following amino acid
sequence:
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DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATG
VPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2)
The term "Abeta antibody A", as used herein, relates to the exemplified Abeta antibody
comprising a heavy chain as defined in SEQ ID NO: 1 and a light chain as defined in SEQ ID
NO: 2.
The term "mono-glycosylated antibody(ies)", as used herein, relates to antibody
molecules comprising an N-glycosylation in one (VH)-region of an individual antibody
molecule, e.g. of an immunoglobulin, e.g. an IgG, e.g. of an IgGl. For example, said "mono-
glycosylated form" comprises a glycosylation on one variable region of the heavy chain e.g.
at position asparagine "Asn 52" of the herein described "Abeta antibody A". This "mono-
glycosylated IgGl -form or mono-glycosylated isoform" may also comprise, as illustrated
herein, the glycosylation in the well conserved glycosylation site in the Fc-part, for example
asparagine Asn 306 in the non-variable Fc-part of the herein exemplified "Abeta antibody
A".
The term "double-glycosylated antibody(ies)" in the meaning of this invention
comprises the herein defined glycosylation on both variable regions of the heavy chain (VH)-
region. Again, this "double glycosylated form", comprises a glycosylation on the variable
region of both heavy chains, e.g. at position asparagine Asn 52 of the herein exemplified
"Abeta antibody A". This "double-glycosylated IgGl -form or double-glycosylated isoform"
may also comprise, as illustrated herein, the glycosylation in the well conserved
glycosylation site in the non-variable/constant Fc-part, in particular on position 306 of the
exemplified "Abeta antibody A". Appended figure 1 illustrates corresponding antibody
molecules.
Antibodies devoid of such a post-translational modification in the variable region, e.g.
in both variable regions of the heavy chain (both (VH)-regions) are, in context of this
invention considered as a "non-glycosylated form", comprising no glycosylation in the
variable region of the heavy chain. Yet, this "non-glycosylated form" may nevertheless
comprise (a) glycosylation(s) in the constant region (C-region) of the antibody, for example,
and most commonly at the well conserved glycosylation site of the Fc-part, in particular the
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asparagine (Asn) 306 in the non- variable/constant Fc-part as defined herein; see also SEQ ID
NO: 1.
The pharmaceutical parenteral formulations of the invention may comprise the
exemplary "Abeta antibody A" as defined herein above and as illustrated in the appended
examples. Accordingly, said pharmaceutical parenteral formulations comprising Abeta
antibody A may comprise mono-glycosylated Abeta antibody A or double-glycosylated
Abeta antibody A or non-glycosylated Abeta antibody A or mixtures thereof as defined
above.
Purification of glycosylation isoforms of recombinantly expressed Abeta antibody
molecules may comprise the steps of:
(1) protein A column purification;
(2) ion exchange column purification, e.g. a cation exchange chromatography;
and, optionally,
(3) size exclusion column purification.
The purification protocol may comprise further steps, like further concentration steps,
e.g. diafiltration or analytical steps, e.g. involving analytical columns. It is also envisaged
and feasible that particular certain steps are repeated (e.g. two ion exchange chromatography
steps may be carried out) or that certain steps (e.g. size exclusion chromatography) may be
omitted.
Protein A is a group specific ligand which binds to the Fc region of most IgGl
isotypes. It is synthesized by some strains of Staphylococcus aureus and can be isolated
therefrom and coupled to chromatographic beads. Several types of gel preparations are
available commercially. An example for a protein A column which may be used is a
MabSelect (Trademark) column. Ideally the column is equilibrated with 25 mM Tris/HCl, 25
mM NaCl, 5 mM EDTA, the cell culture supernatant is loaded onto the column, the column
is washed with 1 M Tris/HCl pH 7,2 and the antibody is eluted at pH 3.2 using 100 mM
acetic acid.
Cation-exchange chromatography exploits interactions between positively charged
groups in a stationary phase and the sample which is in the mobile phase. When a weak
cation exchanger (e.g. CM Toyopearl 650®) is used, the following chromatographic steps are
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performed: After preequilibration with 1 00 mM acetic acid pH 4, loading of Protein A eluate
and washing with 100 mM acetic acid pH 4 the antibody is eluted and fractionated by
applying steps of 250 mM sodium acetate (pH 7.8-8.5) and 500 mM sodium acetate (pH 7.8-
8.5). With the first step a mixture of double-glycosylated isoform fraction and mono-
glycosylated isoform fraction are normally eluted, using the second step the non-glycosylated
isoform fraction is normally eluted.
From a strong cation exchanger (e.g. SP Toyopearl 650) the antibody can be eluted by
salt steps: After equilibration of the column with 50 mM acetic acid pH 5.0, loading the
Protein A eluate with pH 4 the first elution step using 50 mM acetic acid and 210 mM
sodium chloride is performed. Then a second elution step of 50 mM acetic acid and 350 mM
sodium chloride is applied. By the first salt step a mixture of the double-glycosylated isoform
fraction and mono-glycosylated isoform fraction are normally eluted, by the second salt step
the non-glycosylated isoform is normally eluted.
In addition the antibody may also be eluted from a strong cation exchanger column
(e.g. SP-Sepharose®) by a salt gradient: After preequilibration, loading and washing the
column at pH 4.5 a salt gradient is applied from 50 mM MES pH 5.8 to 50 mM MES /l M
sodium chloride pH 5.8. Here the double-glycosylated isoform, mono-glycosylated isoform
and non-glycosylated isoform fractions are normally eluted separately. In the following
double-glycosylated isoform fraction and mono-glycosylated isoform fraction may be pooled
to result in the product pool and/or a desired antibody mixture.
Further purification of the mixture of double- and mono-glycosylated antibody
molecules, e.g. inimunoglobulins, may be performed by size exclusion chromatography. An
example of a useful column is a Superdex 200® column. Examples of running buffers
include histidine/sodium chloride, e.g. 10 mM histidine/125 mM sodium chloride/pH 6, and
phosphate buffered saline (PBS).
Anion exchange chromatography in the flow through mode followed by a
concentration/ diafiltration is an alternative purification step. Q Sepharose® is an example
for a resin for the anion exchange step. For example, the eluate from the SP chromatography
may be threefold diluted with 37,5 mM Tris/HCl pH 7.9 and passed over a Q-Sepharose
column pre-equilibrated with 25 mM Tris/83 mM sodium acetate. The flow through is
collected, adjusted to pH 5.5 and concentrated by ultrafiltration using e.g. a Hydrosart 30
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kD® membrane. In the following the concentrate may be diafiltrated against for example 1 0
volumes of 20 mM histidine/HCl pH 5.5.
As defined above, antibody isoforms may also comprise (a) further glycosylation(s) in
the constant/non-variable part of the antibody molecules, e.g. in the Fc-part of an IgG, e.g. in
the Fc-part in an IgGl. Said glycosylation in the Fc-part relates to a well conserved
glycosylation, being characterized in located on position Asn306 of the heavy chain, e.g., in
accordance with the herein defined SEQ ID NO: 1.
The IgG-Fc region of the antibodies comprised in the formulations of this invention
may be a homodimer comprised of inter-chain disulphide bonded hinge regions, glycosylated
CH2 domains, bearing N-linked oligosaccharide at asparagine 306 (Asn-306) of the CH2 and
non-covalently paired CH3 domains. The oligosaccharide of the glycosylation at Asn-306 is
of the complex biantennary type and may comprise a core heptasaccharide structure with
variable addition of outer arm sugars.
The oligosaccharide influences or determines Fc structure and function (Jefferis (1998)
Immunol Rev. 163, 50-76). Effector functions, numbering particular specific IgG-Fc/effector
ligand interactions have been discussed (Jefferis (2002) Immunol Lett. 82(1-2), 57-65 and
Krapp (2003) J Mol Biol. 325(5), 979-89). This conserved Fc-position Asn-306 corresponds
to "Asn-297" in the Kabat-system (Kabat (1991) Sequences of Proteins of Immunological
Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda MD.)
In a certain embodiment, the formulation of the invention is a liquid or lyophilized
formulation comprising:
- about 1 to about 200 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
- atpH5.5.
In another embodiment, the formulation according to the invention also comprises a
lyophilized formulation comprising:
- 75 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
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- 20 mM L-histidine,
-250 mM Sucrose,
-atpH5.5.
or
-75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
-atpH 5.5.
In yet another embodiment, the formulation according to the invention also comprises
a liquid formulation comprising:
-37.5 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
-10 mM L-histidine,
-125 mM Sucrose,
-atpH5.5.
or
- 37.5 mg/mL Abeta antibody ,
- 0.01% Tween 20 w/v,
- 1 0 mM L-histidine,
-125 mM Sucrose,
-atpH 5.5.
In still another embodiment, the formulation according to the invention also comprises
a lyophilized formulation comprising:
- 1 5 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
-atpH 5.5.
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In still another embodiment, the formulation according to the invention also comprises
a lyophilized formulation comprising:
- 20 mg/mL Abeta antibody ,
- 0.01 1% Tween 20 w/v,
-5.3 mM L-histidine,
- 66.7 mM Sucrose,
atpH 5.5.
In still another embodiment, the formulation according to the invention also comprises
a liquid formulation comprising:
-7.5 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
-250 mM Sucrose,
-atpH5.5;
or
-7.5 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 1 0 mM L-histidine,
-125 mM Sucrose,
atpH5.5.
In a further embodiment, the formulation according to the invention also comprises a
lyophilized formulation comprising:
- 75 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
-atpH 5.5.
or
- 75 mg/mL Abeta antibody ,
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- 0.02% Jween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
-atpH5.5.
In still another embodiment, the formulation according to the invention also comprises
a liquid formulation comprising:
- 37.5 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
-10 mM L-histidine,
-125 mM Trehalose,
-atpH5.5.
or
-37.5 mg/mL Abeta antibody ,
- 0.0 1 % Tween 20 w/v,
-10 mM L-histidine,
-125 mM Trehalose,
-atpH5.5.
In still another embodiment, the formulation according to the invention also comprises
a liquid formulation comprising:
- 75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
-atpH 5.5.
or
- 75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Mannitol,
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-atpH5.5.
or
-75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 140 mM Sodium Chloride,
-at pH 5.5.
or
-150 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
-atpH 5.5.
or
-150 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Mannitol,
-atpH5.5.
or
-150 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
-140 mM Sodium Chloride,
-atpH 5.5.
or
- 1 0 mg/mL Abeta antibody ,
- 0.01% Tween 20 w/v,
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- 20 mM L-histidine,
- 140 mM Sodium chloride,
- atpH5.5
In a preferred embodiment, the formulation according to the invention also comprises a
liquid formulation comprising:
- 1 0 mg/mL Abeta antibody ,
- 0.0 1 % Tween 20 w/v,
- 20 mM L-histidine,
- 140 mM Sodium chloride,
atpH 5.5
In another preferred embodiment, the formulation according to the invention also
comprises a lyophilized formulation comprising:
- 75 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
atpH5.5
In another preferred embodiment, the formulation according to the invention also
comprises a lyophilized formulation comprising:
- 20 mg/mL Abeta antibody ,
- 0.01 1% Tween 20 w/v,
- 5.3 mM L-histidine,
- 66.7 mM Sucrose
atpH 5.5
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FIGURE LEGENDS
Figure 1
Figure 2
Figure 3
Scheme of double-, mono- and non-glycosylated antibody molecules
(immunoglobulins).
Content of monomer as determined by size-exclusion chromatography of Abeta
antibody A formulations after start and incubation at 5°C, 25°C/60%rh and
40°C/75%rh for up to 6 months. Antibody preparations are freeze-dried and
reconstituted to nominal concentration of 75mg/mL.
Content of monomer as determined by size-exclusion chromatography of Abeta
antibody A formulations after start and incubation at 5°C, 25°C/60%rh and
40°C/75%rh for 3 months. Antibody preparations K, L and N are formulated at
75mg/mL, whereas preparations O, P and Q are formulated at 150mg/mL.
EXAMPLES
Liquid and lyophilized drug product formulations for subcutaneous administration
according to the invention were developed as follows:
Preparation of liquid formulations
Abeta antibody comprising a heavy chain as defined in SEQ ID NO: 1 and a light chain
as defined in SEQ ID NO: 2 ("Abeta antibody A" in the context of the present invention) was
prepared and obtained as described in WO 03/070760 and was concentrated by ultrafiltration
to a concentration of approx. 40 to about 200 mg/mL in a 20 mM histidine buffer at a pH of
approx. 5.5. The concentrated solution was then diluted with the formulation buffer
(containing sugar (respectively salt or polyol), surfactant and buffer at a pH of approx. pH
5.5) resulting the anticipated antibody concentration of approx. 7.5mg/mL, 37.5 mg/mL, 75
mg/mL or 150mg/mL formulated in the final bulk composition (e.g. 10 mM L-histidine, 125
mM sucrose, 0.02% Tween 20, at pH 5.5).
Alternatively, Abeta antibody A was buffer-exchanged against a diafiltration buffer
containing the anticipated buffer and sugar composition and concentrated to an antibody
concentration equal or higher than the final concentration of approx. 37.5mg/mL. The
surfactant was added after completion of the ultrafiltration operation as a 100 to 200-fold
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stock solution to the antibody solution. The concentrated antibody solution was adjusted with
a formulation buffer containing the identical excipient composition to the final Abeta
antibody A concentration of approx. 37.5 mg/mL.
All formulations were sterile-filtered through 0.22 urn low protein binding filters and
aseptically filled under nitrogen atmosphere into sterile 6 mL glass vials closed with ETFE
(Copolymer of ethylene and tetrafluoroethylene) -coated rubber stoppers and alucrimp caps.
The fill volume was approx. 2.4 mL. These formulations were stored at different climate
conditions for different intervals of time and stressed by shaking (1 week at a shaking
frequency of 200 min" 1 at 5°C) and freeze-thaw stress methods. The samples were analyzed
before and after applying the stress tests by the analytical methods 1) UV spectrophotometry,
2) Size Exclusion Chromatography (SEC) and 3) nephelometry to determine the turbidity of
the solution.
Preparation of lvophilized formulations and liquid formulations reconstituted
from such lvophilized formulations
Solutions of approx. 37.5 mg/ml "Abeta antibody A" were prepared as described above
for liquid formulations. Any lyophilization method known in the art is intended to be within
the scope of the invention. For example, the lyophilization process used for this study
included the cooling of the formulation from room temperature to approx 5°C (pre-cooling)
and a freezing step to -40°C at a plate cooling rate of approx. l°C/min, followed by a holding
step at -40°C for about 2 hours . The first drying step was performed at a plate temperature of
approx. -25°C and a chamber pressure of approx. 80 jibar for about 62 hours. Subsequently,
the second drying step started with a temperature ramp of 0.2°C / min from -25°C to 25°C,
followed by a holding step at 25°C for at least 5 hours at a chamber pressure of approx. 80
ubar (the applied drying schedule is presented in Table 1 .)
Lyophilization was carried out in an Usifroid SMH-90 LN2 freeze-dryer (Usifroid,
Maurepas, France). All lyophilized cakes in this study had a residual water content of about
0.1 to 1.0% as determined by Karl-Fischer method. The freeze-dried samples were incubated
at different temperatures for different intervals of time.
The lyophilized formulations were reconstituted to a final volume of 1 .2 mL with water
for injection (WFI) yielding an isotonic formulation with an antibody concentration of
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approx. 75 mg/mL and a viscosity of less than 3 mPa-s. The reconstitution time of the freeze-
dried cakes was about 2 to 4min. Analysis of the reconstituted samples was either performed
immediately after reconstitution, or after a 24 hour incubation period of the reconstituted
liquid sample at 25 °C.
The samples were analyzed by 1) UV spectrophotometry, 2) determination of the
reconstitution time, 3) Size Exclusion Chromatography (SEC) and 4) method of
nephelometry to determine the turbidity of the solution.
Size Exclusion Chromatography (SEC) was used to detect soluble high molecular
weight species (aggregates) and low molecular weight hydrolysis products (LMW) in the
formulations. The method was performed on a Merck Hitachi 7000 HPLC instrument
equipped with a Tosohaas TSK G3000 SWXL column. Intact monomer, aggregates and
hydrolysis products are separated by an isocratic elution profile, using 0.2M K2HPO4 /
0.25M KCL, pH 7.0 as mobile phase, and were detected at a wavelength of 280nm.
UV spectroscopy, used for determination of protein content, was performed on a
Varian Cary Bio UV spectrophotometer at 280 nm. Neat protein samples were diluted to
approx. 0.5 mg/mL with 20 mM L-histidine, pH 5.5. The protein concentration was
calculated according equation 1 .
The protein concentration was measured with a precision of ±10%. The UV light absorption
at 280 nm was corrected for light scattering at 320 nm and multiplied with the dilution
factor, which was determined from the weighed masses and densities of the neat sample and
the dilution buffer. The numerator was divided by the product of the cuvette's path length d
and the extinction coefficient e.
Clarity and the degree of opalescence were measured as Formazin Turbidity Units
(FTU) by the method of nephelometry. The neat sample was transferred into all mm
diameter clear-glass tube and placed into a HACH 2 100 AN turbidimeter.
Equation 1 :
Protein content =
,4(280) - AQ20) x dil. factor
WO 2008/071394 PCT/EP2007/010825
28
Table 1 Freeze-drving Cycle type I
Step
Shelf
temperature
(°C)
Ramp Rate
(°C/min)
Hold time
(min)
Vacuum Set
point
(u-bar)
Pre-cooling
5°C
0.0
60
Freezing
-40°C
1.0
150
Primary Drying
-25°C
0.5
3700
80
Secondary
Drying
+25°C
0.2
300
80
WO 2008/071394
PCT/EP2007/010825
Table 2 Compositions of "Abeta antibody A" drug product formulations according to
the invention
Formulation
Composition
(Stability data in Table)
Lyophilized Formulations
Formulation A
75 mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Sucrose,
0.04% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone, after
reconst.
(*)(mg/mL)
Size Exclusion - HPLC
Turbidity
after
reconst.
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
72.8
1.9
96.1
2.0
5.4
24 hat
25°C after
reconst.
74.8
1.9
96.0
2.1
5.3
1 month
at 2-8°C
74.5
1.7
95.8
2.5
5.4
3 months
at 2-8°C
74.2
2.0
95.9
2.1
5.6
6 months
at 2-8°C
n.d.
2.0
96.0
2.0
n.d.
6 months at
25°C/60%rh
n.d
2.3
95.7
2.0
n.d
6 months at
40°C/75%rh
n.d.
3.2
94.8
2.0
n.d.
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Formulation B
75 mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Sucrose,
0.02% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone, after
reconst. (*)
(mg/mL)
Size Exclusion - HPLC
Turbidity
after
reconst.
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
74.9
1.9
96.1
2.0
5.3
24 hat
25°C after
reconst.
73.8
1.9
96.1
2.0
5.2
1 month
at 2-8°C
• 74.3
1.7
95.9
2.4
5.4
3 months
at 2-8°C
73.9
2.0
95.9
2.1
6.0
6 months
at 2-8°C
n.d.
2.0
96.0
2.0
n.d.
6 months at
25°C/60%rh
n.d
2.3
95.7
2.0
n.d
6 months at
40°C/75%rh
n.d.
3.2
94.8
2.0
n.d.
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Formulation C
75 mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Trehalose,
0.04% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone, after
reconst. (*)
(mg/mL)
Size Exclusion - HPLC
Turbidity
after
reconst.
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
74.4
2.0
96.1
2.0 5.3
24 hat
25°C after
reconst.
73.6
2.6
96.0
2.1
5.1
1 month
at 2-8°C
72.7
1.7
95.7-95.9
2.4
5.3
3 months
at 2-8°C
72.5
2.0
95.9
2.1
5.2
6 months
at 2-8°C
n.d.
2.0
96.0
2.0
n.d.
6 months at
25°C/60%rh
n.d
2.6
95.4
2.0
n.d
6 months at
40°C/75%rh
n.d.
4.2
93.8
2.0
n.d.
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Formulation D
75 mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Trehalose,
0.02% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone, after
reconst. (j)
(mg/mL)
Size Exclusion - HPLC
Turbidity
after
reconst.
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
73.6
2.0
96.1
2.0
5.2
24 hat
25°C after
reconst.
72.8
2.0
96.0
2.0
5.6
1 month
at 2-8°C
72.9
1.8
95.8
2.4
5.1
3 months
at 2-8°C
73.4
2.0
95.9
2.1
5.5
6 months
at 2-8°C
n.d.
2.0
96.0
2.0
n.d.
6 months at
25°C/60%rh
n.d
2.6
95.4
2.0
n.d.
6 months at
40°C/75%rh
n.d.
4.2
93.8
2.0
n.d.
Formulation E
1 5 mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Sucrose,
0.04% polysorbate 20,
atpH 5.5
(*) taking into account the analytical precision and slight variability of reconstitution.
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Liquid Formulations
Formulation F
Storage at 2-8°C
37.5mg/mL Abeta antibody A,
1 0 mM L-histidine,
125 mM Sucrose,
0.02% polysorbate 20,
at pH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
36.7
1.8
96.2
2.0
3.5
1 week
shaking
36.8
1.8
96.2
2.0
3.6
3 months
37.8
1.8
96.1
2.1
3.4
Formulation G
Storage at 2-8°C
37.5mg/mL Abeta antibody A,
1 0 mM L-histidine,
125 mM Sucrose,
0.01% polysorbate 20,
at pH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
36.8
1.8
96.2
2.0
3.3
1 week
shaking
36.8
1.8
96.3
1.9
3.6
3 months
37.8
1.8
96.1
2.1
3.9
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Formulation H
Storage at 2-8°C
37.5mg/mL Abeta antibody A,
10 mM L-histidine,
125 mM Trehalose,
0.02% polysorbate 20,
atpH5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
36.6
1.8
96.2
2.0
3.6
1 week
shaking.
36.6
1.8
96.2
2.0
3.4
3 months
37.7
1.8
96.1
2.1
4.2
Formulation I
Storage at 2-8 °C
37.5mg/mL Abeta antibody A,
1 0 mM L-histidine,
1 25 mM Trehalose,
0.01% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
36.6
1.8
96.2
2.0
3.5
1 week
shaking.
36.4
1.8
96.2
2.0
3.5
3 months
37.8
1.8
96.1
2.1
3.7
Formulation J
7.5 mg/mL Abeta antibody A,
1 0 mM L-histidine,
125 mM Sucrose,
0.02% polysorbate 20,
at pH 5.5
WO 2008/071394
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Formulation K
75mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Trehalose,
0.02% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
75.3
0.9
98.5
0.6
5.0
1 week
shaking,
at 2-8°C
77.0
0.8
98.6
0.6
4.9
3 months
at 2-8°C
70.5
0.8
98.6
0.6
5.2
3 months at
25°C/60%rh
72.0
0.9
98.3
0.8
8.1
3 months at
40°C/75%rh
69.1
1.5
95.7
2.9
6.9
Formulation L
75mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Mannitol,
0.02% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
76.6
0.9
98.5
0.6
5.7
1 week
shaking,
at 2-8°C
77.4
0.8
98.6
0.6
5.5
3 months
at 2-8°C
81.1
0.8
98.6
0.6
5.7
3 months at
25°C/60%rh
72.0
0.9
98.3
0.8
8.4
3 months at
40°C/75%rh
72.9
1.4
95.8
2.8
8.6
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Formulation M
Storage at 2-8°C
1 Omg/mL Abeta antibody A,
20 mM L-histidine,
140 mM Sodium chloride,
0.01%polysorbate 20,
atpH 5.5
Size Exclusion - HPLC
Turbidity
(FTU)
Timepoint
Protein
cone.
(mg/mL)
HMW (%)
Monomer
(%)
LMW (%)
Initial
9.7
0.7
98.1
1.2
3.7
1 week
shaking
9.7
0.7
98.0
1.3
3.8
3 months
9.6
0.7
98.0
1.3
3.7
Formulation N
75mg/mL Abeta antibody A,
20 mM L-histidine,
140 mM Sodium Chloride,
0.02% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
73.9
1.0
98.5
0.6
17.5
1 week
shaking,
at 2-8°C
80.0
0.9
98.5
0.6
18.7
3 months
at 2-8°C
74.5
1.0
98.5
0.6
18.6
3 months at
25°C/60%rh
72.1
1.1
98.1
0.8
19.4
3 months at
40°C/75%rh
70.4
2.1
94.9
3.0
n.d.
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Formulation O
150mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Trehalose,
0.02% polysorbate 20,
at pH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
143.7
1.0
98.5
0.6
5.7
1 week
shaking,
at 2-8°C
151.9
1.0
98.5
0.6
5.0
3 months
at 2-8°C
138.1
1.1
98.3
0.6
5.5
3 months at
25°C/60%rh
134.5
1.5
97.8
0.8
7.3
3 months at
40°C/75%rh
141.7
3.0
94.3
2.8
6.2
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Formulation P
150mg/mL Abeta antibody A,
20 mM L-histidine,
250 mM Mannitol,
0.02% polysorbate 20,
atpH 5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
146.4
1.0
98.5
0.6
5.8
1 week
shaking,
at 2-8°C
153.4
1.0
98.5
0.6
5.3
3 months
at 2-8°C
141.1
1.1
98.4
0.6
5.9
3 months at
25°C/60%rh
146.7
1.5
97.8
0.8
7.1
3 months at
40°C/75%rh
138.1
2.8
94.4
2.8
7.1
Formulation Q
150mg/mL Abeta antibody A,
20 mM L-histidine,
140 mM Sodium Chloride,
0.02% polysorbate 20,
atpH5.5
Timepoint
Protein
cone.
(mg/mL)
Size Exclusion - HPLC
Turbidity
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
150.8
1.0
98.5
0.6
18.0
1 week
shaking,
at 2-8°C
158.3
1.0
98.5
0.6
19.0
3 months
at 2-8°C
136.0
1.1
98.3
0.6
17.5
3 months at
25°C/60%rh
148.5
1.6
97.7
0.8
19.0
3 months at
40°C/75%rh
144.4
3.4
93.8
2.8
19.6
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lyophilized Formulation
Formulation R
20 mg/mL Abeta antibody A,
5.3 mM L-histidine,
66.7 mM Sucrose,
0.01 1% polysorbate 20,
at pH 5.5
Timepoint
Protein
cone, after
reconst. (*)
(mg/mL)
Size Exclusion - HPLC
Turbidity
after
reconst.
(FTU)
HMW (%)
Monomer
(%)
LMW (%)
Initial
19.4
0.8
99.1
0.1
1.4
1 month
at 2-8°C
19.6
0.8
99.1
0.1
1.5
3 months
at 2-8°C
19.4
0.8
99.1
0.1
1.5
3 months at
25°C/60%rh
19.5
1.0
98.9
0.1
1.6
3 months at
40°C/75%rh
19.5
1.7
98.2
0.1
1.6
(*) taking into account the analytical precision and slight variability of reconstitution.
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40
Claims
1. A stable pharmaceutical parenteral Abeta antibody formulation comprising:
about 1 to about 250 mg/raL Abeta antibody;
about 0.001 to about 1% of at least one surfactant;
about 1 to about 1 00 mM of a buffer;
optionally about 10 to about 500 mM of a stabilizer and/or about 5 to about 500 mM of
a tonicity agent;
at a pH of about 4.0 to about 7.0.
2. The formulation according to claim 1 wherein it is a liquid formulation.
3. The formulation according to claim 1 wherein it is a lyophilized formulation.
4. The formulation according to claim 1 wherein it is a liquid formulation reconstituted
from a lyophilized formulation.
5. The formulation according to any one of claims 1 to 4, wherein the Abeta antibody
concentration is of about 1 to about 200 mg/mL.
6. The formulation according to claim 5 wherein the Abeta antibody concentration is of
about 50 mg/mL to about 200 mg/mL.
7. The formulation according to claim 6 wherein the Abeta antibody concentration is of
about 1 50 mg/mL to about 200 mg/mL.
8. The formulation according to any one of claims 1 to 7 wherein the stabilizer is
present in the formulation in an amount of about 10 to about 300mM.
9. The formulation according to claim 1 to 7, wherein the stabilizer is present in the
formulation in an amount of about 100 to about 300mM
10. The formulation according to any one of claims 1 to 9, wherein the stabilizer is
selected from the group consisting of sugars, amino acids, polyols, surfactants, antioxidants,
preservatives, cyclodextrines, in particular hydroxypropyl-P-cyclodextrine, sulfobutylethyl-
(3-cyclodextrin and p-cyclodextrin, polyethylenglycols, in particular PEG 3000, 3350, 4000
WO 2008/071394
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41
and 6000, albumin, human serum albumin (HSA), bovines serum albumin (BSA), salts in
particular sodium chloride, magnesium chloride, calcium chloride and chelators, in particular
EDTA.
11. The formulation according to any one of claims 1 to 10, wherein the stabilizer is a
lyoprotectant.
12. The formulation according to claim 1 1, wherein the lyoprotectant is selected from
the group consisting of sugars, amino acids, polyols and sugar alcohols.
13 The formulation according to claim 12, wherein the lyoprotectant is selected from
the group consisting of trehalose, sucrose, mannitol, lactose, glucose, mannose, maltose,
galactose, fructose, sorbose, raffinose, glucosamine, N-Methylglucosamine ("Meglumine"),
galactosamine, neuraminic acid and arginine.
14. The formulation according to any one of claims 1 to 13 wherein the surfactant is
present in the formulation in an amount of about 0.005 to about 0.1 % w/v.
15. The formulation according to claim 14, wherein the surfactant is present in the
formulation in an amount of about 0.01% to about 0.04%w/v
16. The formulation according to any one of claims 1 to 15 wherein the surfactant is
selected from the group consisting of polyoxyethylensorbitan fatty acid esters,
polyoxyethylene alkyl ethers, alky lphenylpolyoxy ethylene ethers, polyoxyethylene-
polyoxypropylene copolymer and sodium dodecyl sulphate
17. The formulation according to claim 16, wherein the surfactant is selected from the
group of polyoxyethylene sorbitan monolaureate and polyoxyethylene sorbitan monooleate,
poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407,
polyoxyethylene (23) lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene (10)
oleyl ether and polyoxyethylene (20) oleyl ether, and octyl phenol ethoxylate (7.5), octyl
phenol ethoxylate (9.5), and octyl phenol ethoxylate (102).
18. The formulation according to claim 17, wherein the surfactant is selected from the
group containing polyoxyethylene sorbitan monolaureate and polyoxyethylene sorbitan
monooleate
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42
19. The formulation according to any one of claims 1 to 18 wherein the buffer is
present in the formulation in an amount of about ImM to about 100 mM.
20. The formulation according to claim 15, wherein the buffer is present in the
formulation in an amount of about 5 mM to about 50 mM.
21. The formulation according to claim 20, wherein the buffer is present in the
formulation in an amount of about 10 to about 20 mM.
22. The formulation according to any one of claims 1 to 21 wherein the buffer is
selected from the group consisting of histidine-buffers, citrate-buffers, succinate-buffers,
acetate-buffers and phosphate-buffers.
23. The formulation according to claim 22 wherein the buffer comprises L-histidine or
mixtures of L-histidine with L-histidine hydrochloride.
24. The formulation according to any one of claims 1 to 23, wherein the pH is about
4.0 to about 7.0.
25. The formulation according to claim 24, wherein the pH is about 5.0 to about 6.0.
26. The formulation according to claim 25, wherein the pH is about 5.5.
27. The formulation according to any one of claims 1 to 26, which comprises one or
more tonicity agents.
28. The formulation according to any one of claims 1 to 27, wherein the tonicity agent
is present in the formulation in an amount of about 5 mM to about 500 mM.
29. The formulation according to any one of claims 1 to 28, wherein the tonicity agents
are selected from the group consisting of sodium chloride, potassium chloride, glycerin,
amino acids, sugars, as well as combinations thereof.
30. The formulation according to any one of claim 1 to 29, which can be administered
by intravenous (i.v.) or subcutaneous (s.c.) or any other parenteral administration.
31. The liquid formulation of claim 2 which comprises:
- about 1 to about 200 mg/mL Abeta antibody,
- 0.04% Tween 20 w/v,
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43
- 20 mM L-histidine,
- 250 mM Sucrose,
- atpH5.5;
or
- 37.5 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
-10 mM L-histidine,
-125 mM Sucrose,
-atpH5.5;
or
-37.5 mg/mL Abeta antibody ,
-0.01% Tween 20 w/v,
-10 mM L-histidine,
-125 mM Sucrose,
-atpH 5.5;
or
- 7.5 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
-250 mM Sucrose,
-atpH 5.5;
or
- 7.5 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
-10 mM L-histidine,
-125 mM Sucrose,
-atpH5.5;
or
-37.5 mg/mL Abeta antibody ,
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44
- 0.02% Tween 20 w/v,
-10 mM L-histidine,
- 125 mM Trehalose,
-atpH 5.5;
or
- 37.5 mg/mL Abeta antibody ,
- 0.01% Tween 20 w/v,
- 1 0 mM L-histidine,
-125 mM Trehalose,
-atpH5.5.
or
- 75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
-atpH5.5.
or
- 75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Mannitol,
-atpH5.5.
or
- 75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 140 mM Sodium chloride,
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45
-atpH5.5.
or
- 150 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
-atpH 5.5.
or
-150 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
-250 mM Mannitol,
-atpH 5.5.
or
-150 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
-140 mM Sodium chloride,
- at pH 5.5;
or
- 1 0 mg/mL Abeta antibody ,
- 0.01 % Tween 20 w/v,
- 20 mM L-histidine,
- 140 mM Sodium chloride,
-atpH 5.5.
32. The lyophilized formulation of claim 3 which comprises:
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46
- about 1 to about 200 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
- atpH5.5;
or
-75 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
-atpH5.5;
or
- 75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
- at pH 5.5;
or
- 1 5 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
-atpH5.5;
or
- 75 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
-atpH5.5.
WO 2008/071394
PCT/EP2007/010825
47
or
- 75 mg/mL Abeta antibody ,
- 0.02% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Trehalose,
atpH 5.5
or
- 20 mg/mL Abeta antibody ,
- 0.01 1% Tween 20 w/v,
-5.3 mM L-histidine,
- 66.7 mM Sucrose,
atpH5.5.
33. The liquid formulation of claim 2 or 31 which comprises:
- 1 0 mg/mL Abeta antibody ,
- 0.0 1 % Tween 20 w/v,
- 20 mM L-histidine,
- 140 mM Sodium chloride,
atpH 5.5.
34. The lyophilized formulation of claim 3 or 32 which comprises:
- 75 mg/mL Abeta antibody ,
- 0.04% Tween 20 w/v,
- 20 mM L-histidine,
- 250 mM Sucrose,
atpH 5.5.
35. The lyophilized formulation of claim 3 or 32 which comprises:
- 20 mg/mL Abeta antibody ,
- 0.01 1% Tween 20 w/v,
WO 2008/071394
PCT/EP2007/010825
48
-5.3 mM L-histidine,
- 66.7 mM Sucrose,
atpH 5.5.
36. The formulation according to claims 1 to 35, wherein the Abeta antibody comprises
at least one antigen binding site comprising a glycosylated asparagine (Asn) in the variable
region of the heavy chain (V H ).
37. The formulation according to claim 1 to 36, wherein the Abeta antibody is a
defined mixture of
(a) Abeta antibody, wherein one of the antigen binding sites comprises a glycosylated
asparagine (Asn) in the variable region of the heavy chain (Vh); and
(b) Abeta antibody, wherein both antigen binding sites comprise a glycosylated asparagine
(Asn) in the variable region of the heavy chain (V H );
and which is free of or comprises to a very low extent Abeta antibody, wherein none of the
antigen binding site comprises a glycosylated asparagine (Asn) in the variable region of the
heavy chain (Vh).
38. The formulation according to claim 36 or 37, wherein the glycosylated asparagine
(Asn) in the variable region of the heavy chain (V H ) is a glycosylated asparagine (Asn) in the
CDR-2 region of the heavy chain (V H ).
39. The formulation according to claims 1 to 38, wherein the Abeta antibody comprises
a heavy chain as defined in SEQ ID NO: 1 and a light chain as defined in SEQ ID NO: 2.
40. Use of a formulation according to any one of claims 1 to 39 for the preparation of a
medicament useful for treating Alzheimer's disease.
41. The invention as described hereinabove.
WO 2008/071394
PCT/EP2007/010825
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INTERNATIONAL SEARCH REPORT
International application No
PCT/EP2007/0 10825
A. CLASSIFICATION OF SUBJECT MATTER
INV. C07K16/18 A61K39/395
A61K9/00
A61K47/00
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
A61K C07K •
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practical, search terms used)
EPO-Internal , BIOSIS, EMBASE, WPI Data
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
WO 2006/081587 A . (WYETH CORP [US]; LUISI
DONNA [US]; WARNE NICHOLAS W [US]; KANTOR
ANGEL) 3 August 2006 ■ (2006-08-03)
page 4, paragraph 6 - page 5, paragraph 1
page 5, paragraph 2.
page 35, paragraph 3
page 4, paragraph 4
page 5, paragraph 3
page 5, paragraph 4
page 6, paragraph 3 -
page 10, paragraph 1
page 13, paragraph 3 - page 14, paragraph
1
page 17, paragraph
page 36, paragraph
page 7, paragraph 1
page 37,
page 37,
2
page 39,
paragraph
paragraph
paragraph 4
- page 38, paragraph
1-35,40
Further documents are listed in the continuation of Box C.
See patent family annex.
* Special categories of cited docu ments :
"A" document defining the general state of the art which is not
considered to be of particular relevance
"E" earlier document but published on or afterthe international
filing date
"L" document which may throw doubts on priority claim(s) or
which is cited to establish the publication date of another
citation or other special reason (as specified)
"O" document referring to an oral disclosure, use, exhibition or
other means
"P" document published prior to the international filing date but
later than the priority date claimed
"T" later document published afterthe international filing date
or priority date and not in conflict with the application but
cited to understand the principle or theory underlying the
invention
"X" document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document Is taken alone
"Y" document of particular relevance; the claimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.
■&" document member of the same patent family
Date of the actual completion of the international search
28 March 2008
Date of mailing of the international search report
10/04/2008
Name and mailing address of the ISA/
European Patent Office, P.B. 5818 Patentlaan 2
NL - 2280 HV Rijswijk
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl.
Fax: (+31-70) 340-3016
Authorized officer
Irion, Andrea
Form PCT/ISA/210 (second sheet) (April 2005)
INTERNATIONAL SEARCH REPORT
International application No
PCT/EP2007/010825
C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
Y
X
Y
Y
P,X
A
A
P,A
page 40, paragraph 4
WO 2006/083689 A (NEURALAB LTD; WYETH CORP
[US]; LUISI DONNA [US]; WARNE NICHOLAS W
[US] ) 10 August 2006 (2006-08-10)
page 8, lines 26,27
page 20, lines 9-22
line 20 - page 22, line 4
lines 26,27
1 i ne 28 - page 42 ,
line 29 - page 23,
lines 3-7
line 7 - page 42, line 12
line 29
lines 4-28
line 30 - page 44, line 28
line 29 - page 46, line 17
1 ine 16
36-39
1-35,40
page 21,
page 22,
page 41,
page 22,
page 23,
page 34,
page 42,
page 43,
page 43,
page 45,
page 3.
line 1
line 2
W0 03/070760 A (HOFFMANN LA ROCHE [CH];
MORPHOSYS AG [DE]; BARDROFF MICHAEL [DE];
BOHR) 28 August 2003 (2003-08-28)
cited in the application
the whole document
sequences 89,91
36-39
36-39
W0 2007/068429 A (HOFFMANN LA ROCHE [CH]:
LOETSCHER HANSRUEDI [CH]; HUBER WALTER
[CH] ; S) 21 June 2007 (2007-06-21)
the whole document
page 42 - page 47
1-40
W0 03/009817 A (PROTEIN DESIGN LABS INC
[US] ; KAISHEVA ELIZABET A [US] ;
FLORES-NATE AL)
6 February 2003 (2003-02-06)
the whole document
1-40
•US 2004/197324 Al (LIU JUN [US] ET AL)
7 October 2004 (2004-10-07)
the whole document
US 2003/202972 Al (ANDYA JAMES [US] ET AL
ANDYA JAMES [US] ET AL)
30 October 2003 (2003-10-30)
the whole document
W0 2007/110339 A (HOFFMANN LA ROCHE [CH].
GROSSMANN ADELBERT [DE]; MAHLER
HANNS-CHRISTIA)
4 October 2007 (2007-10-04)
the whole document
1-40
1-40
1-40
Form PCT/ISA/210 (continuation oJ second sheet) (April 2005)
International Application No. PCT/EP2007 /010825
FURTHER INFORMATION CONTINUED FROM PCT71SA/ 21 0
Continuation of Box 1 1. 2
CI aims Nos . : 41
Claim 41 does not contain any technical feature which may define the
subject-matter. Therefore, claim 41 is so unclear that a meaningful
search is impossible (Article 6 PCT).
The applicant's attention is drawn to the fact that claims relating to
inventions in respect of which no international search report has been
established need not be the subject of an international preliminary
examination (Rule 66.1(e) PCT). The applicant is advised that the EPO
policy when acting as an International Preliminary Examining Authority is
normally not to carry out a prel i mi nary examination on matter which has
not been searched. This is the case irrespective of whether or not the
claims are amended following receipt of the search report or during any
Chapter II procedure. If the application proceeds into the regional phase
before the EPO, the applicant is reminded that a search may be carried
out during examination before the EPO (see EPO Guideline C-VI, 8.2),
should the problems which led to the Article 17(2)PCT declaration be
overcome.
INTERNATIONAL SEARCH REPORT
International application No.
PCT/EP2007/01Q825
Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)
This international search report has not been established in respect of certain claims under Article 1 7(2)(a) for the following reasons:
*
1- | I Claims Has.:
because they relate to subject matter not required to be searched by this Authority, namely:
2. * Claims Nos.: 41
because they relate to parts of the International application that do not comply with the prescribed requirements to such
an extent that no meaningful international search can be carried out, specifically:
see FURTHER INFORMATION sheet PCT/ISA/210
3. | | Claims Nos.:
because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).
Box Mo. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)
This Internationa] Searching Authority found multiple inventions in this international application, as follows:
1 . | I As all required additional search fees were timely paid by the applicant, this international search report covers allsearchable
' ' claims.
2. | | As all searchable claims could be searched without effort Justifying an additional fees, this Authority did not invite payment of
additional 'fees.
3. I As only some of the required additional search fees were timely paid by the applicant, this international search reportcovers
' ' only those claims for which fees were paid, specifically claims Nos.:
4. | | No required additional search fees were timely paid by the applicant. Consequently, this international search report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
Remark on Protest I ~ ' |The additional search fees were accompanied by the applicant's protest and, where applicable, the
' 'payment of a protest fee.
□ The additional search fees were accompanied by the applicant's protest but the applicable protest
ffifi was not raid within thf* Hmfi limit Rnenifieri in ths invitation.
1 fee was not paid within the time limit specified in the invitation.
| | No protest accompanied the payment of additional search fees.
Form PCT/ISA/210 (continuation of first sheet (2)) (April 2005)
INTERNATIONAL SEARCH REPORT
Information on patent family members
International application No
PCT/EP2007/010825
Patent document
cited in search report
Publication
date
Patent family
member(s) '
Publication
date
WO 2006081587 A
03-08-2006
W0 2006083689 A
10-08-2006
AR
052469
Al
21-
■03-
-2007
AU
2006207901
Al
03-
-08-
-2006
CA
2595380
Al
03-
-08-
-2006
CN
101111264
A
23-
•01-
-2008
EP
•I n A *i /i r /*
1841456
A2
10-
■10-
-2007
KR
o n r\ "7 a 1 r\ ~~i r\ ~r r~\
20070107079
A
06-
•11-
-2007
NO
20073666
B
25-
•10-
-2007
UY
29350
Al
31-
-08-
-2006
AR
052198
Al
07-
•03-
-2007
AU
2006211184
Al
10-
-08-
-2006
CA
2593122
Al
10-
-08-
-2006
EP
1853310
A2
14-
-11-
-2007
KR
20070108193
A
08-
-11-
-2007
NO
20073305
B
24-
-08-
-2007
UY
29351
Al
31-
-08-
-2006
WO 03070760 A 28-08-2003
AU
2003218995
Al
09-
-09-
-2003
BR .
0307837
A
07-
-12-
-2004
CA
2477012
Al
28-
-08-
-2003
CN
1630665
A
22-
-06-
-2005
HR
20040712
A2
30-
-06-
-2005
JP
2005527199
T
15-
-09-
-2005
MX
PA04008077
A
14-
-12-
-2005
US
2005169925
Al
04-
-08-
-2005
ZA
200406604
A
14-
-06-
-2005
WO
2007068429
A
21-
-06-
-2007
AR
057233
Al
21-11-2007
UY
30003
Al
31-08-2007
WO
03009817
A
06-
-02-
-2003
AU
2002324556
Al
17-02-2003
CA
2454587
Al
06-02-2003
EP
1409018
A2
21-04-2004
JP
2004538287
T
24-12-2004
MX
PA04000747
A
08-07-2004
US
2004197324
Al
07-
-10-
-2004
US
2005158303
Al
21-07-2005
US.
2007053900
Al
08-03-2007
US
2003202972
Al
30-
-10-
-2003
NONE
WO 2007110339 A 04-10-2007 NONE
Form PCT/ISA/S1 0 (patent family annex) (April 2005)
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